Process for producing amino acids by fermentation

ABSTRACT

L-lysine, L-aspartic acid, L-alanine, L-valine, L-leucine and Larginine are produced by culturing Protaminobacter thiaminophagus ATCC 21927 in a nutrient medium containing methanol as the carbon source under aerobic conditions.

"United States Patent Nakayama et al.

[111 3,907,641 [451 Sept. 23, 1975 PROCESS FOR PRODUCING AMINO ACIDS BY FERMENTATION Inventors: Kiyoshi Nakayama, Sagamihara;

Mamoru Kohata, Kawasaki; Yoshitake Tan'aka, Machida; Tadaaki Nomura, Tokyo; Ryoichi Katsumata, Maebashi, all of Japan Assignee: Kyowa Hakko Kogyo Co., Ltd.,

Tokyo, Japan Filed: Apr. 5, 1974 Appl. No.: 458,423

Foreign Application Priority Data \[56] References Cited UNITED STATES PATENTS 3,663,370 5/1972 Kono et a1. 195/49 OTHER PUBLlCATlONS Chem. Abstracts, 72: P 131095u.

Primary ExaminerAlvin E. Tanenholtz Assistant ExaminerThomas G. Wiseman Attorney, Agent, or Firm-Fitzpatrick, Celia, Harper & Scinto [57] ABSTRACT L-lysine, L'aspartic acid, L-alanine, L-valine, L- leucine and L-arginine are produced by culturing Protaminobacter thiaminophagus ATCC 21927 in a nutrient medium containing methanol as the carbon source under aerobic conditions.

5 Claims, No Drawings PROCESS FOR PRODUCING AMINO ACIDS BY FERMENTATION BACKGROUND OF THE INVENTION alanine, L-valirie, L-leucine and L-arginine by culturing a newly obtained mutant, Protaminobacter-thiaminplzagus [(-2 l 7, ATCC 21927 in a nutrient medium containing methanol as a source 'of carbon.-

Amino acids including those above-mentioned;are well known to be useful in various aspects and are, therefore, in an ever increasing demand.

Variousprocesses have been known in the art toproiduce amino acids by culturing either wild or mutant strains of microorganisms in a medium containing carbohydrates, hydrocarbons and organic acids as a source of carbon.

In these years, many attempts have been made on the use of alcohols such as methanol and ethanol for a fer- ,mentation raw material. Particularly, much attention has been paid on the use of methanol sincemethanol is not only available at a low price but also easy to handie in a fermentation procedure. U.S. Pat. Nos. 3,707,441 and 3,708,395 suggest the use of alcohols for the fermentative production "of L- lysine, but neither'a practical nor concrete process is disclosed therein. I

Reviewing the prior art on the practical production of amino acids from alcohols by fermentation, U.S. Pat.

No. 3,595,751 discloses a production of L-lysine from ethanol by a strain of the genera Corynebacterium, Brevibacterium, Arthrobacter, Bacillus and Nocardia. U.S. Pat. No. 3,663,370 is directed to the production of L-glutarnic acid from methanol by certain strains of microor ganisms of the genera M ethanomonas, Protaminibacter and Microcyclus. U.K. Pat. No. 1,210,770 teaches a culturing of yeasts in a medium containing methanol to produce microbial cells as well as certain amino acids. Further, Japanese Patent Publication No. ,25,273/70 discloses an accumulation of amino acids by strains of the genera Aclzromobacter and Pseudomonas using methanol as a carbon source. However, known amino acids-producers from methanol are not satisfactory in view of either productivity or the variety of amino acids to be produced, and therefore, improved producers are still desired.

The present inventors have carried out an induction of mutation on various methanol-utilizing microorganiisms to obtain such improved producers of amino acids, and as a result they have screened a mutant strain K-2l7 derived from Protaminobacter thiaminop/zagus ATCC 21371 which is disclosed in the above mentioned U.S. Pat. No. 3,663,370 including its taxonomical characteristics.

The newly isolated mutant, Proraminobacter tlu'aminuphagus K-217 is capable of accumulating L- ,lysine, L-aspartic acid, L-alanine, L-valine, L-leucine and L-arginine in a considerable amount when cultured in a medium containing methanol as a source of carbon.

Protaminobacter thiaminop/mgus K-2l7 has been deposited with American Type Culture Collection, Rockville, Maryland, U.S.A., accorded an accession number ATCC 21927 and is freelyavailable therefrom public. Y n

DESCRIPTION OF THE INVENTION The present invention provides a process for produc' ing various amino acids, i.e. L-lysine, L-aspartic acid, L-alanine, L-valine, L-leucine and L-arginine by culturing Protaminobacter thiaminophagus, K-2l7. ATCC 21927 in a nutrient medium containing methanol as the main carbon source.

As the fermentation medium employed in the present process for culturing the ATCC 21927 strain, any synthetic or natural medium can be employed, so long as it contains methanol as a carbon source, nitrogen, sources, inorganic materials, vitamins and growthpromoting nutrients atproper concentrations.

Methanol to be used as a main carbon source someto the times causes the growth inhibition of microorganisms when present ata high concentration the medium.

Generally it is desirable that the concentration of methanol'in the medium; be maintained below about '3%(v/v). Good results can be obtained when a medium initially having a low concentratiom for example, 0.5

3%(v/v) of methanol is used and culturing is carried out while f eeding methanol to the medium continuously, in an amount of 0.3 0.6%( v/v)'b ased on the volume of the medium per hour, or intermittently in an amount of 0.5 2%(v/v) based on the volume of the medium at each feeding, as methanol is consumed by the microorganism.

As the nitrogen source, ammonium salts such as amdium as the nitrogen source. Additionally, as inorganic materials, potassium phosphates, magnesium sulfate, iron and manganese salts may be used. The strain of the present invention, i.e. Protaminobacter tlziaminophagus K-2l7, ATCC 21927 has the requirement for thiamine. Therefore, thiamine or a natural product containing thiamine must be added to the medium.

Culturing is carried out under aerobic conditions at 20 to 40C for 2 to 5 days. In order to obtain a high yield of the product, it is desirable that the pH of the medium be maintained at 4 to 9, preferably, at around neutral during culturing. The pH may be adjusted with calcium carbonate, various buffer solutions or alkaline solutions.

After the completion of culturing, the microbial cells are removed from the culture broth by filtration. The produced amino acids are recovered from the resulting filtrate by any of the well known methods, such as ion exchange resin treatment, crystalization by concentration, etc.

The following example is presented for illustrating the present invention.

EXAMPLE 1 In this example, Protaminobacter thiaminophagus K-2l7 (FERM-P No. 2021) ATCC -2l927 is used. This strain is inoculated in ml of a seed medium having the following composition in a test tube and cultured at 30C for hours with shaking.

' Methanol 20 ml (NH-02804 g KH P 2 g K HPO 7 g 1 MgSO .7H O 1 0.5 g FeSO .7H O 10 mg MnSO .4H O 8 mg Thiamine hydrochloride 1 mg Biotin l0 ug Yeast extract 0.l-g Phenol red (pH-indicator) l0 mg Water to make upthe total volume (pH: 7.2)..

The resulting seed'culture is inoculated into 10 ml portions of a fermentation medium havingthe same composition asthe seed mediumin test tubes ina ratio of 1 ml. 'Culturing is carried out at 30C for 96 hours with shaking. 24 hours, 48 hours and 72 hours after the start of the culturing, 2% of methanol is added to each culture broth total amount of the added methanol: 6%). The pH of the culture broth is adjusted about neutrality using 14% aqueous ammonia. After the completion of culturing, 0.2 mg/ml of L-aspartic acid, 0.8 mg/ml of L-alanine, 1.0 mg/ml of L-valine, 1.2 mg/ml of 1.- l'eucine, 0.7 mg/ml of L-arginine and 0.3 mg/ml of L- lysine (asL-lysine hydrochloride) are accumulated in from one liter of the culture is 0.14 g,.0.48 g, 0.6 g, 0.72

g, 0.49 g and 0.21 g, respectively.

What is claimed is:

1. A process for producing L-lysine, L-aspartic acid,.

L-alanine, L-valine, L-leucine and L-arginine which comprises culturing protaminobacter thiaminophagus ATCC 21927 in a nutrient medium containing methan'ol as the carbon source under aerobic conditions, accumulating L-lysine, L-aspartic acid, L-alanine, L- valine, L-leucineand L-arginine in the medium and isolating at least oneof said amino acids.

2. A process of claim 1, wherein said culturing is carried out at 20 to 40C.

3. A process according to claim 1 wherein said culturing is carried out at about neutral pH.

4. A process according to claim 1 wherein said methanol is fed to said culture medium continuously in an amount of 0.3 to 0.6% by volume based upon the volume of the culture medium during said culturing step.

5. A process according to claim 1 wherein said methanol is fed to said culture medium intermittently in an amount of 0.5 to 2% by volume based upon the volume of said culture medium during said culturing step. 

1. A PROCESS FOR PRODUCING L-LYSINE, L-ASPARTIC ACID, ALANINE, L-VALINE, L-LEUCINE AND L-ARGININE WHICH COMPRISING CULTURING PROTAMINOBACTER THIAMINOPHAGUS ATCC 21927 IN A NUTRIENT MEDIUM CONTAINING METHANOL AS THE CARBON SOURCE UNDER AEROBIC CONDITIONS, ACCUMULATING L-LYSINE, L-ASPARTIC ACID, L-ALANINE, L-VALINE, L-LEUCINE AND L-ARGININE IN THE MEDIUM AND ISOLATING AT LEAST ONE OF SAID AMINO ACIDS.
 2. A process of claim 1, wherein said culturing is carried out at 20* to 40*C.
 3. A process according to claim 1 wherein said culturing is carried out at about neutral pH.
 4. A process according to claim 1 wherein said methanol is fed to said culture medium continuously in an amount of 0.3 to 0.6% by volume based upon the volume of the culture medium during said culturing step.
 5. A process according to claim 1 wherein said methanol is fed to said culture medium intermittently in an amount of 0.5 to 2% by volume based upon the volume of said culture medium during said culturing step. 